Nicotine abuse either from tobacco cigarette or e-cigarette smoking is one of the most important risk factors in development of cardiovascular disease. Recent studies in women and animal models indicate that cigarette smoking or nicotine use by mothers during pregnancy increases the risk of hypertension and cardiovascular diseases in offspring. Oxidative stress has been implicated in the etiology of many diseases with substantial public health impact including chronic cardiovascular disorders and preeclampsia. Our recent studies in pregnant rats have demonstrated that perinatal nicotine exposure causes a development of vascular dysfunctional phenotype associated with an increased vascular specific NADPH oxidase (NOX2) gene expression and reactive oxygen species (ROS) production in adult offspring. However, the molecular mechanisms underlying nicotine-mediated programming of vascular oxidative stress are unclear. In addition, whether the heightened oxidative stress contributes to increase risk of vascular dysfunction in the adulthood remains undetermined. Thus, the proposed studies will test the central hypothesis that perinatal nicotine exposure epigenetically programs of NOX2 gene through DNA hypomethylation mechanism, resulting in increased vascular oxidative stress and development of vascular dysfunction phenotype in offspring. In this proposed study we will focus on two specific aims using a well-established pregnant rat model and offspring. Aim 1 will determine whether perinatal nicotine causes CpG hypomethylation at specific transcription factor binding sites in NOX2 promoter, resulting in increased its promoter activity. Aim 2 will determine whether treatment with methyl donor reverses nicotine-mediated hypomethylation and NOX over-expression. Furthermore, whether inhibition of NOX2 rescues nicotine-mediated excess ROS and reverses nicotine- mediated heightened vascular contractility and blood pressure response in offspring. The proposed studies will provide important and novel insights into the epigenetic mechanism of DNA methylation in up-regulation of NOX gene and NOX-derived ROS in vasculature. The outcomes will also provide novel evidence that fetal programming of vascular oxidative stress is the molecular mechanistic linker between nicotine and development of vascular dysfunction phenotype, which may lead to novel diagnostic tools and therapeutic approaches to prevent cardiovascular disease-induced by nicotine abuse in pregnancy.